Specific mechanisms prevent non-aminoacylated tRNA from binding ribosomes
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Peer-reviewed literature demonstrates that translational mechanisms and fidelity processes prevent the inappropriate binding of uncharged or non-cognate tRNAs to the ribosome.
To synthesize a protein, a ribosome moves along a messenger RNA (mRNA), reads it codon by codon, and takes up the corresponding ternary complexes which consist of aminoacylated transfer RNAs (aa-tRNAs), elongation factor Tu (EF-Tu), and GTP. During this process of translation elongation, the ribosome proceeds with a codon-specific rate. Here, we present a general theoretical framework to calculate codon-specific elongation rates and error frequencies based on tRNA concentrations and codon usages. Our theory takes three important aspects of in-vivo translation elongation into account. First, non-cognate, near-cognate and cognate ternary complexes compete for the binding sites on the ribosomes. Second, the corresponding binding rates are determined by the concentrations of free ternary complexes, which must be distinguished from the total tRNA concentrations as measured in vivo. Third, for each tRNA species, the difference between total tRNA and ternary complex concentration depends on the codon usages of the corresponding cognate and near-cognate codons. Furthermore, we apply our theory to two alternative pathways for tRNA release from the ribosomal E site and show how the mechanism of tRNA release influences the concentrations of free ternary complexes and thus the codon-specific elongation rates. Using a recently introduced method to determine kinetic rates of in-vivo translation from in-vitro data, we compute elongation rates for all codons in Escherichia coli. We show that for some tRNA species only a few tRNA molecules are part of ternary complexes and, thus, available for the translating ribosomes. In addition, we find that codon-specific elongation rates strongly depend on the overall codon usage in the cell, which could be altered experimentally by overexpression of individual genes.
We utilized the nedicistrovirus (NediV) intergenic region (IGR) internal ribosomal entry site (IRES)-mediated, initiation factor-independent translation initiation system and determined high-resolution structures of 80S ribosome complexes with the NediV IRES in various functional states, including binary complexes, aminoacyl-transfer RNA (tRNA)-bound complexes, and complexes with elongation factor eEF2. In binary complexes, the NediV IRES primarily occupies the ribosomal P site, exhibiting conformational flexibility and engaging the ribosome at multiple interaction sites. Upon translocation, the IRES undergoes structural rearrangements, including destabilization of its PKI domain, facilitating the transition to canonical elongation. Crucially, we captured an eEF2-bound complex, along with an eEF1A-bound failed decoding complex featuring a mismatched tRNA, the latter representing the first instance of a canonical elongation complex visualized in the presence of a natural, hydrolysable nucleotide and without the addition of any trapping agents. These findings provide a comprehensive structural overview of IGR IRES-mediated translation initiation and its transition to elongation, revealing key mechanistic details of viral translation and proofreading.
The mechanism of suppression by ribosomal mutations is proposed to consist of the release of this growth inhibition by the reduction of the rate of polypeptide synthesis, which would keep amino acid incorporation from exceeding the slow charging of tRNA and thus exhausting the pool of charged tRNA. In the suppressor strains, therefore, growth at the semi-restrictive temperature is no longer limited by the aminoacylation of tRNA but by the translational process at the mutated ribosome. This influence of the ribosomal mutation on the speed of translation could be directly or indirectly coupled with an effect on translational fidelity resulting in the prevention of the binding of uncharged or non-cognate charged tRNA or in the tighter binding of peptidyl-tRNA when cognate aminoacyl-tRNA is limiting. Published in Molecular & general genetics : MGG (1976)
GTP-binding protein 1 (GTPBP1) is a widespread translational GTPase closely related to elongation factor eEF1A. The loss of GTPBP1 leads to neurodevelopmental and neurodegenerative disorders in animals. Although linked to translation and quality control mechanisms, GTPBP1 molecular functions remain largely obscure. Similarly to eEF1A, GTPBP1 delivers aminoacyl-tRNA to the ribosome, but the ensuing GTPBP1-mediated elongation is slow. Here, using cryo-EM of mammalian 80S ribosomal complexes bound to GTPBP1 and aa-tRNA with GTP or the non-hydrolysable analog GDPCP, we show that the distinct GTPBP1 architecture and interactions with tRNA underlie slow GTPBP1 dissociation after GTP hydrolysis, resulting in delayed tRNA accommodation. Slow dissociation correlates with an extended proofreading stage and higher accuracy of GTPBP1-mediated decoding, potentially allowing GTPBP1 to elicit its putative quality control functions. GTPBP1 visualization provides the foundation for mapping and elucidating GTPBP1 mutations associated with human diseases.
Effect of initiation factor 3 binding on the 30S ribosomal subunits of Escherichia coli. Under certain conditions, initiation factor 3 (IF-3) can cause the release of aminoacyl-tRNA bound to 30S ribosomal subunits of E. coli. It is shown that this IF-3-induced aminoacyl-tRNA release cannot be attributed to either nucleolytic attack or competition between IF-3 and aminoacyl-tRNA for the same ribosomal binding site. It was found that the 30S-aminoacyl-tRNA-codon complexes formed in the absence of IF-3 are intrinsically different from those prepared in the presence of IF-3. In the absence of IF-3, the ribosomal binding of aminoacyl-tRNA is a virtually irreversible process, since the bound aminoacyl-tRNA can neither be spontaneously released upon dilution nor exchanged for unbound aminoacyl-tRNA. In the presence of IF-3, the binding of one molecule of IF-3 per 30S ribosome renders the binding of aminoacyl-tRNA reversible upon dilution and promotes exchange between bound and unbound aminoacyl-tRNA. It is suggested that this difference is due to a conformational transition of the 30S ribosomal subunit induced by the binding of IF-3.
Translation is the process by which the genetic information contained in mRNA is used to determine the sequential order of amino acids in a protein. Experimental measurements have suggested that, overall, an amino acid is misincoroprated at the rate of about 1 in every 10,000 codons. This high level of fidelity is ensured by various types of proofreading and editing mechanisms used throughout protein biosynthesis. The maturation of tRNAs and mRNAs is monitored, as is the identity of amino acids attached to tRNAs. Accuracy is further enhanced during the selection of aminoacyl-tRNAs on the ribosome and their base pairing with mRNA. Recent studies have begun to reveal the molecular basis of quality control processes which are essential for faithful translation. Aminoacyl-tRNA synthetases are the only components of the gene expression machinery that function on the interface between the nucleic acids and proteins. These enzymes catalyze esterification of tRNAs with cognate amino acids and display high level of substrate specificity. During substrate selection tRNA first binds the aminoacyl-tRNA synthetase, either as a free enzyme or as an enzyme – aminoacyl-adenylate complex. The recognition is governed by sequence-specific protein-RNA interactions. Then, this transient protein RNA-complex catalyzes attachment of the amino acid to the 3´-terminal adenosine of the tRNA. While the differences of the side chains of amino acids are often sufficient to allow their specific bindi
Translation is the process by which the genetic information contained in mRNA is used to determine the sequential order of amino acids in a protein. Experimental measurements have suggested that, overall, an amino acid is misincoroprated at the rate of about 1 in every 10, 000 codons. This high level of fidelity is ensured by various types of proofreading and editing mechanisms used throughout protein biosynthesis. The maturation of tRNAs and mRNAs is monitored, as is the identity of amino acids attached to tRNAs. Accuracy is further enhanced during the selection of aminoacyl-tRNAs on the ribosome and their base pairing with mRNA. Recent studies have begun to reveal the molecular basis of quality control processes which are essential for faithful translation. Aminoacyl-tRNA synthetases are the only components of the gene expression machinery that function on the interface between the nucleic acids and proteins. These enzymes catalyze esterification of tRNAs with cognate amino acids and display high level of substrate specificity. During substrate selection tRNA first binds the aminoacyl-tRNA synthetase, either as a free enzyme or as an enzyme – aminoacyl-adenylate complex. The recognition is governed by sequence-specific protein-RNA interactions. Then, this transient protein RNA-complex catalyzes attachment of the amino acid to the 3´-terminal adenosine of the tRNA. While the differences of the side chains of amino acids are often sufficient to allow their specific binding, s
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